We seek to gain a better understanding of the role of the antibody repertoire as a whole in providing heterosubtypic immunity to influenza virus. We have found that we can group of antigen binding sites into categories defined by specific physical-chemical properties. We have shown that some categories are preferred at key stages of life, whereas others appear to be actively selected against, especially in young adults who typically are more resistant to heterosubtypic infection than aged individuals. Preliminary studies indicate that immunoglobulins using 'disfavored'categories of antigen binding sites can be found in aged individuals. Because the general principles of antibody repertoire control appear common to human and mouse, in order to study the role of repertoire control in heterosubtypic immunity we created mice whose repertoire is limited to one of these highly disfavored categories. We previously used these mice to show that when antibodies bearing antigen binding sites belonging to normally preferred categories were completely replaced by antibodies using a disfavored category, protection against heterosubtypic challenge with an H1N1 strain was severely compromised. However, these studies did not answer the question of whether it was the absence of the normally preferred repertoire or the presence of sequences belonging to a disfavored category that had impaired heterosubtypic immunity. In the present proposal, we test the hypothesis that the presence of disfavored immunoglobulin can have a dominant negative effect on protection against heterosubtypic infection. This is a key issue because, as noted above, the composition of the repertoire can change with age or disease. Mice heterozygous for altered IgH alleles containing mutations in the DH locus produce a normal repertoire from the normal allele, and disfavored antibodies from the gene targeted allele. These mice will be used to test whether the presence of these disfavored antibodies is sufficient to increase morbidity and mortality after mice are immunized with either an H3N2 or an H1N1 strain, and then challenged with homologous or heterologous viruses. The increase in morbidity and mortality will confirm that control of the global composition of the antibody repertoire is essential for properly protective immune responses to influenza virus. Two potential mechanisms that could underlie this increases susceptibility to infection will then be tested. Mice will be injected with sera or infused with bone marrow mononuclear cells from naove or immunized mice, and then challenged with homologous or heterologous viruses. An increase in morbidity after administration with the sera will point to a direct effect of soluble disfavored antibody. An increase in morbidity only after transfer of cells will suggest a direct role of immunoglobulin as a B cell receptor, perhaps by altering antigen presentation to T cells. We will then titer the effect to determine the threshold beyond which heterosubtypic immunity is impaired. Coupled with new means to evaluate repertoire diversity, this information will then be used to inform susceptibility to influenza virus infections and to guide vaccination and treatment strategies. PUBLIC HEALTH RELEVANCE: We seek to understand how the composition of the totality of antibodies produced by the body may affect protection against influenza virus. We plan to use this information both to identify individuals at risk for greater morbidity and mortality, and to guide vaccination and treatment strategies.